Ligand-binding properties of estrogen receptor proteins after interaction with surface-immobilized Zn(II) ions: evidence for localized surface interactions and minimal conformational changes.
The site- or domain-specific immobilization of steroid receptor proteins with preserved structure and function would facilitate the identification and purification of receptor-associated regulatory components and nucleic acids. We have demonstrated previously that restricted surface regions of the estrogen receptor protein contain high affinity binding sites for immobilized Zn(II) ions. Possible conformational changes in receptor at the stationary phase immobilized metal ion interface were evaluated by monitoring alterations in the equilibrium dissociation constant (Kd) for [3H]estradiol. Soluble estrogen receptor proteins (unliganded) present in immature calf uterine cytosol were immobilized via surface-exposed Zn(II)-binding sites to beads of agarose derivatized with iminodiacetate (IDA)-Zn(II) ions. The IDA-Zn(II) bound receptor was incubated with increasing concentrations of [3H]estradiol (0.01-20 nM) in the presence and absence of unlabeled competitor (diethylstilbestrol) to determine the level of specific hormone binding. Steroid-binding experiments were performed in parallel with identical aliquots of soluble receptor. Analyses of the equilibrium binding data revealed the presence of a single class of high-affinity (Kd = 2.44 +/- 1.5 nM, n = 10) steroid-binding sites which were only marginally affected by receptor immobilization via surface-exposed Zn(II) bindings sites (Kd = 2.58 +/- 0.56 nM, n = 4). These data are consistent with the location of surface accessible Zn(II) binding site(s) on the receptor at or near the DNA binding domain which, upon occupancy, do not influence the steroid binding domain.(ABSTRACT TRUNCATED AT 250 WORDS)
Hutchens
T W
TW
Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030.
Li
C M
CM
D004958:Estradiol / Q000378:metabolism; D008024:Ligands; D011487:Protein Conformation; D011960:Receptors, Estrogen / Q000737:chemistry / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
D008024:Ligands; D011960:Receptors, Estrogen; D004958:Estradiol; D015032:Zinc